I. Field of the Invention
The present invention pertains to multi-dose formulations of erythropoietin (hereinafter “EPO”), comprising a particularly advantageous preservative or combination of preservatives. Specifically, the present invention pertains to the use of the preservatives benzethonium chloride, phenoxyethanol and phenylethyl alcohol, alone or in combination, in multi-dose EPO formulations. The invention disclosed herein also relates to a method of treating a patient with an EPO-containing composition comprising benzethonium chloride, phenoxyethanol and/or phenylethyl alcohol. The present invention further relates to a pharmaceutical carrier composition; a vial for containing a composition; and a method of inhibiting microbial growth in a solution; wherein all compositions or solutions comprise EPO and one or more of the preservatives benzethonium chloride, phenoxyethanol and phenylethyl alcohol.
II. Background of the Invention
Sterility is one of the most important characteristics of parenteral products. For parenteral products that are sterilized and intended for single dose injection, maintenance of sterility is a function of both the method of sterilization and the integrity of the packaging system. For parenteral products that are intended for multiple dosing, antimicrobial agents must be added to the product formulation to protect the product from accidental microbial contamination during its storage and/or use.
Stable protein-containing multi-dose pharmaceutical formulations are viewed by the pharmaceutical industry as particularly advantageous and commercially attractive. Multi-dose formulations are generally, though not always, contained in vials (multi-dose containers) that allow for the extraction of partial amounts of the formulation at various times. This type of system is desirable as it allows multiple doses to be obtained from a single container, and allows for more controlled administration of the pharmaceutical composition as the formulation may be withdrawn and administered in any partial amount.
The nature of the use of multi-dose formulations imposes special requirements on the formulation. For example, maintenance of the sterility of the composition is particularly challenging given the many opportunities for introduction of microorganisms and other contaminants into the formulations. Repeated introduction of foreign elements, for example, needles, into the multi-dose container after formulation creates a likelihood of introducing microorganisms into the container. Additionally and alternatively, microorganisms may be introduced during filling of the containers, or during reconstitution of the formulations after lyophilization and prior to administration. The extended periods of time over which the container may be stored—especially during multiple introductions of foreign elements, and/or after contaminants may have been introduced, demands that the formulation contain special additives to insure the sterility of the contents.
To insure that multi-dose formulations maintain optimally sterile properties, the United States Food and Drug Administration (FDA) and regulatory agencies in other jurisdictions require that all multi-dose formulations contain preservatives to prevent the growth of, or to affirmatively kill, any microorganisms that may be introduced into the formulations. Given the inherent instability of proteins, and their tendency to interact adversely with preservative compounds, the development of protein containing multi-dose formulations has proven particularly difficult. Possible adverse interactions between preservatives and proteins include the degradation of the protein, especially when stored for extended periods of time; inactivation of the protein; formation of protein aggregates; and other interactions that inactivate the formulation or make administration of the formulation to humans, particularly by infusion, injection or other parenteral administration, difficult, painful or otherwise undesirable.
Additionally, preservatives themselves are noted for causing acute adverse reactions, such as allergic reactions, in humans upon parenteral administration. Ideally, the preservative contained in the multi-dose protein pharmaceutical composition should be effective in low concentration against a wide variety of micro organisms, soluble in the formulation, non-toxic, compatible and non-reactive with the protein, active with long term stability, and non-reactive with components of the container or closure system.
Sandeep Nema et al. published lists of various excipients that have been included in the formulation of injectable products marketed in the USA. The antimicrobial preservatives listed in this review article are included in Table 1:
TABLE 1ANTIMICROBIAL PRESERVATIVESPreservativeFrequencyRangeBenzalkonium chloride10.02% w/vBenzethonium chloride40.01%Benzyl alcohol740.75-5%Chlorobutanol170.25-0.5%m-cresol30.1-0.3%Myristyl gamma-picolinium20.0195-0.169%chlorideParaben methyl500.05-0.18%Paraben propyl400.01-0.1%Phenol480.2-0.5%2-Phenoxyethanol30.50%Phenyl mercuric nitrate30.001%Thimerosal460.003-0.01%
EPO is a glycoprotein that functions to stimulate the production of hemoglobin and erythrocytes in bone marrow. It is produced in the kidney, and is widely used as a treatment for anemia caused by a variety of conditions, including, for example, renal failure. The amino acid sequence and general glycosylation patterns of EPO are known in the art. See, for example, Miyaka et al. and U.S. Pat. No. 4,703,008. Isolation and Purification of EPO, from human tissues or fluids, has been described by Miyake et al.
The nucleic acid sequence encoding the protein, isolation of this sequence, and production of the protein by traditional recombination methods are also known in the art. See, for example, U.S. Pat. No. 4,703,008 to Lin, describing the nucleic acid sequence encoding EPO; U.S. Pat. No. 4,337,513 to Sugimoto et al., describing the use of lymphoblastoid cells to produce EPO; and Sherwood et al., describing production of EPO by a human renal carcinoma cell line. Additionally, production, isolation and purification of the protein is also achievable by gene-activation, or homologous recombination, followed by well-known isolation and purification techniques.
Development of EPO-containing multi-dose formulations has proven particularly difficult by virtue of the particular instability displayed by EPO, and its tendency to readily interact with common pharmaceutical ingredients. U.S. Pat. No. 4,806,524. Attempts to develop multi-dose EPO formulations have tried to circumvent these problems by maintaining the formulations at a low pH, or by including various amino acid constructs, two approaches thought to assist in the stabilization of the EPO protein, or by developing lyophilized forms in which the preservative sublimes from the formulation before administration. U.S. Pat. No. 5,503,827 (the '827 patent) to Woog.
Stable, sterile multi-dose EPO-containing pharmaceutical formulations are few. They include those formulations disclosed in the '827 patent. The '827 reference discloses and specifically claims chloretone (chlorbutanol, 1,1,1-trichloro-2-methyl-2-propanol), benzalkonium chloride or benzyl alcohol as preservatives. Woog specifically notes the particular difficulty of providing a multi-dose EPO formulation in which the allergy rates are reduced, and promotes the use of the specifically claimed preservatives as especially advantageous in that regard. This reference further stresses that due to the tendency of preservatives to degrade and proteins to be inactivated when combined, it is most desirable to minimize contact between the preservative and the protein. The '827 patent further discloses the use of several amino acid constructs and other additives thought necessary to stabilize EPO in solution. Finally, The '827 patent discloses, in a most preferred embodiment that any preservative used in the initial formulation is sublimed away upon lyophilization of the composition. Then, upon reconstitution, additional preservative selected from the group disclosed (chloretone) as defined, benzalkonium chloride and benzyl alcohol, may be introduced, but the injectable, reconstituted solution should be used within 30 days.
Another example of an EPO-containing multi-dose formulation is described in U.S. Pat. No. 5,661,125 (the '125 patent). This patent explicitly acknowledges and affirms other references stating that EPO “is an instable substance especially in solution form” and “when combined with known stabilizers, the resulting stability of the EPO is varied and unpredictable.” This reference then goes on to show and claim the specific use of benzyl alcohol, a paraben and/or a phenol or a combination of these as a preservative in EPO-containing solutions. Further attesting to the difficulty of discerning compatible and advantageous preservatives for use in EPO-containing multi-dose formulations, this reference states:                “ . . . nothing specific can be derived from the use of preservatives with other proteins that would suggest any particular preserved formulation for erythropoietin. See, e.g., Geigert, J., ‘Overview of the Stability and Handling of Recombinant Protein Drugs,’ Journal of Parenteral Science & Technology, Vol. 43(5):220-224 (1989)”.        
Accordingly, there remains a need for an EPO-containing, preserved, multi-dose pharmaceutical formulation that: (1) maintains the stability of the protein component and the composition over an extended shelf life of the product; (2) maintains the sterility of the formulation and meets the United States, European and Japanese Pharmacopia criteria for preservative challenge testing; (3) is safe in the concentrations used; and (4) is administrable—by any parenteral or oral route—in a manner that is effective, and minimizes pain and the chance of adverse reaction, for example, allergic reaction, in the patient.